FOOD ODOR, VISUAL DANGER STIMULUS, AND RETRIEVAL OF AN AVERSIVE MEMORY TRIGGER HEAT SHOCK PROTEIN HSP70 EXPRESSION IN THE OLFACTORY LOBE OF THE CRAB CHASMAGNATHUS GRANULATUS L. FRENKEL, a1 B. DIMANT, a L. D. SUÁREZ, a E. L. PORTIANSKY b AND A. DELORENZI a * a Laboratorio de Neurobiología de la Memoria, Dto. Fisiología y Bi- ología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IFIBYNE-CONICET, Argentina b Laboratorio de Análisis de Imágenes, Facultad de Ciencias Veteri- narias, Universidad Nacional de La Plata, Argentina Abstract—Although some of the neuronal substrates that support memory process have been shown in optic ganglia, the brain areas activated by memory process are still un- known in crustaceans. Heat shock proteins (HSPs) are syn- thesized in the CNS not only in response to traumas but also after changes in metabolic activity triggered by the process- ing of different types of sensory information. Indeed, the expression of citosolic/nuclear forms of HSP70 (HSC/HSP70) has been repeatedly used as a marker for increases in neural metabolic activity in several processes, including psycho- physiological stress, fear conditioning, and spatial learning in vertebrates. Previously, we have shown that, in the crab Chasmagnathus, two different environmental challenges, wa- ter deprivation and heat shock, trigger a rise in the number of glomeruli of the olfactory lobes (OLs) expressing HSC/ HSP70. In this study, we initially performed a morphometric analysis and identified a total of 154 glomeruli in each OL of Chasmagnathus. Here, we found that crabs exposed to food odor stimuli also showed a significant rise in the number of olfactory glomeruli expressing HSC/HSP70. In the crab Chas- magnathus, a powerful memory paradigm based on a change in its defensive strategy against a visual danger stimulus (VDS) has been extensively studied. Remarkably, the iterative presentation of a VDS caused an increase as well. This in- crease was triggered in animals visually stimulated using protocols that either build up a long-term memory or gener- ate only short-term habituation. Besides, memory reactiva- tion was sufficient to trigger the increase in HSC/HSP70 ex- pression in the OL. Present and previous results strongly suggest that, directly or indirectly, an increase in arousal is a sufficient condition to bring about an increase in HSC/HSP70 expression in the OL of Chasmagnathus. © 2011 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: heat shock proteins, memory, crustaceans, olfac- tory lobes, visual stimulus, arousal. Decapod crustaceans are useful models to investigate central brain functions such as information processing and memory formation. Although some of the neuronal sub- strates that support memory in decapods have been shown in optic ganglia (Tomsic et al., 2009). Specific brain areas activated by either training protocols or retrieval processes are still unknown. An approach to this issue is the use of markers in metabolic activity. In this sense, the expression of heat shock proteins (HSPs) has been re- peatedly used in different species (Ambrosini et al., 2005; Fukudo et al., 1997, 1999; Pizarro et al., 2003). HSPs are constitutively expressed polypeptides that play a role in fundamental functions, most notably as molecular chaper- ones. They participate in protein assembly, secretion, traf- ficking, degradation, and in the regulation of transcription factors and protein kinases (Bukau et al., 2006; Feder and Hofmann, 1999; Parsell and Lindquist, 1993; Sørensen and Loeschcke, 2007). Particularly, the HSP70 family rep- resents one of the largest stress protein families with re- lated members distributed throughout the cells that ex- press multiple and related forms of HSP70, including the cytosolic/nuclear HSP70 proteins HSC70 and HSP70 (HSC/HSP70) (Bukau et al., 2006). Since HSC/HSP70 are synthesized in the postsynaptic structure of most neuronal cells, an increase in their expression levels can be used as a marker for the increase in metabolic neural activity in specifics brain areas, including areas engaged in memory processing and/or psychophysiological stress (Ambrosini et al., 2005; Fukudo et al., 1997, 1999; Pizarro et al., 2003; Suzuki et al., 1999). In crustaceans, heat shock and osmotic and environ- mental stress significantly increase HSP expression in several tissues, including the CNS (Cimino et al., 2002; Chang, 2005; Frenkel et al., 2008; Ravaux et al., 2003; Selvakumar and Geraldine, 2005; Tanguay et al., 2004). In addition, HSC/HSP70 is also involved in the defense strat- egy of Daphnia magna to cope with predator stress (Pi- janowska and Kloc, 2004). We have recently shown that a significant increase in HSC/HSP70 expression is induced under two different environmental challenges in specific brain areas of the crab Chasmagnathus (Frenkel et al., 2008). This euryhaline and semiterrestrial crab inhabits brackish waters and is usually confronted with water de- privation, a natural situation that triggers a series of ho- 1 Present address: Laboratorio de Genética del Comportamiento, Fun- dación Instituto Leloir-IIBBA-CONICET, Argentina. Av. Patricias Ar- gentinas 435-Ciudad de Buenos Aires. (C1405BWE). *Corresponding author. Tel: +54-11-4576-3348; fax: +54-11-4576- 3447. E-mail address: delorenzi@fbmc.fcen.uba.ar (A. Delorenzi). Abbreviations: AC, active control protocol; CSM, context-signal mem- ory; FO, food odor; HSC/HSP70, citosolic/nuclear forms of HSP70; HSPs, heat shock proteins; NC, novel context; NO, no odor; NV, naive; OL, olfactory lobe; PBS, phosphate-buffered saline; PC, passive con- trol; ST, strong training protocol; TC, training context; VDS, visual danger stimulus. Neuroscience 201 (2012) 239–251 0306-4522/12 $36.00 © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.10.052 239